The present invention concerns a method for the quantification of methylated cytosine positions in DNA. 5-methylcytosine is the most commonly modified base in the DNA of eukaryotic cells. It plays an important biological roll in transcriptional regulation, genetic imprinting, and tumorogenesis among other things (for overview: Millar et al.: Five not four: History and significance of the fifth base. In: The Epigenome, S. Beck and A. Olek (eds.), Wiley-VCH Verlag Weinheim 2003, S. 3-20). The identification of 5-methylcytosine is particularly of considerable interest for cancer diagnosis. Detection of methylcytosine is, however, difficult, since cytosine and methylcytosine exhibit the same base-pairing behavior. Conventional DNA-analysis methods based on hybridization are, thus, not applicable. The current methods for methylation analysis operate based on two distinct principles. In one case, methylation-specific restriction enzymes are used, and in another, selective chemical conversion takes place of unmethylated cytosine into uracil (so-called: bisulfite-treatment, see also: DE 101 54 317 A1; DE 100 29 915 A1). The enzymatic or chemically treated DNA can be then in most cases amplified and is analyzed using different methods (for an overview: WO 02/072880 P 1 ff; Fraga and Estella: DNA methylation: a profile of methods and applications. Biotechniques. 2002 September; 33(3): 632, 634, 636-49.). For sensitive analysis, the chemically treated DNA is typically amplified using a PCR method. A selective amplification only of the methylated (or in the reverse approach: unmethylated) DNA can be ensured through the use of methylation specific primers or blockers (so-called methylation sensitive PCR/MSP or “Heavy Methyl method”, compare: Herman et al.: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 1996 Sep. 3; 93(18): 9821-6 Cottrell et al.: A real-time PCR assay for DNA-methylation using methylation-specific blockers. Nucl. Acids. Res. 2004 32: e10). On the other hand, it is also possible to first amplify the DNA in a methylation-specific manner and then to analyze the amplificates using methylation-specific probes (for overview: Trinh et al.: DNA methylation analysis by MethyLight technology. Methods. 2001 December; 25(4):456-62). The so-called PCR methods are also applicable as real-time PCR variations. This permits the detection of the methylation status directly during the course of the PCR without the necessity of a subsequent analysis of the product (“MethyLight”—WO00/70090; U.S. Pat. No. 6,331,393; Trinh et al. 2001, a.a.o.).
A quantification of the degree of methylation is necessary for several applications: for instance, for classification of tumors, for prognostic statements, or for the predictions of pharmaceutical side-effects. There are diverse methods known for the quantification of the degree of methylation. In part, an amplification, thereby, is first performed when using for instance, Ms-SNuPE, hybridization on microarrays, hybridization assays in solution or directly in bisulfite sequencing (for overview: Fraga and Estella 2002, a.a.o.). A problem exists within this “end point analysis” and that is, among other things, the amplification can occur non-uniformly due to product constraints, enzyme instability, and decreases in concentration of reaction components. A correlation between the amount of amplificate and the amount of DNA inserted is not always a given. The quantification is thereby error-prone (compare: Kains: The PCR plateau phase—towards an understanding of its limitations. Biochem. Biophys. Acta 1494 (2000) 23-27). Threshold-value analysis based on real-time PCR determines the quantity of an amplificate not at the end of the amplification but in the exponential phase of the amplification. It is assumed in this method that the amplification efficiency is constant in the exponential phase. The so-called threshold value Ct is the number of PCR cycles required before the signal in the exponential phase of the amplification is for the first time greater than the background noise. The absolute quantification is carried out then by comparing the Ct values of the examined DNA with the Ct values of the standards (compare: Trinh et al. 2001, a.a.o.; Lehmann et al.: Quantitative assessment of promoter hypermethylation during breast cancer development. Am J Pathol. 2002 February; 160(2):605-12). A problem, however, exists with the Ct analysis and that is, with high DNA concentrations only a low resolution can be achieved. The same is true when high degrees of methylation are to be investigated using PMR values (compare to PMR values: Eads et al., CANCER RESEARCH 61, 3410-3418, Apr. 15, 2001.). Additionally, this type of Ct analysis is also necessary for the amplification of a reference gene such as the β-actin gene (compare: Trinh et al 2001, a.a.o.).
Recently, a method for the quantification of methylation analysis has been described where the bisulfite-converted DNA is amplified and detected using both methylation-specific, real-time probes (“QM”-Assay). Thereby, one of the probes is specific for the methylation state, while the other probe is specific for the unmethylated state. Both the probes carry different fluorescent dyes. Within particular PCR cycles, a quantification of the degree of methylation can then be performed based upon, for example, the ratio of signal intensities of both probes or the Cts of the fluorescence channels (compae: European patent application: 04 090 213.2; Lehmann and Kreipe: Real-time PCR-based assay for quantitative determination of methylation status. Methods Mol Biol 2004; 287:207-18; Zeschnigk et al.: A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus. Nucleic Acids Res. 2004 Sep. 7; 32(16):e125.)
In the following is another real-time PCR method described for the quantification of methylated DNA. Here, the bisulfite-treated DNA is likewise amplified and using both real-time probes, detected. In contrast to the method described above, here, however, only one of the probes is specific for the methylated (or the unmethylated) state. The other probe is methylation-specific. With help from both these probes, it is possible to carry out a simple quantification of cytosine methylation according to the invention.
A principle difference exists between the method according to the invention and the well-known Lightcycler method as given in the following: In the Lightcycler method, two oligonucleotide probes are employed, which hybridize to the amplificate spatially near each other. Through an energetic interaction (FRET) between both the probes, a generated fluorescent signal is subsequently detected. In the Lightcycler method, both the probes can only be detected together and not indepently of each other. In contrast, both probes, in the method according to the invention, are tagged with a dye, which permits the independent detection of each probe. As a result, two signals with two different expression qualities are generated: in that, one signal represents the total DNA, and the other signal is specific for the methylated/unmethylated DNA. The degree of methylation can be determined then from the ratio of both signals at the examined position. In an especially preferred embodiment of the method according to the invention, elements from the Lightcycler and the Taqman technologies are combined. This allows for a very efficient quantification.
Due to the known special biological and medical significances of cytosine methylation and due to the abovementioned disadvantages of the state of the art, there exists a great technical need for the development of an efficient method for the quantification of methylation analysis. The method according to the invention makes such a method available and with that, demonstrates an important technical improvement.